Entering edit mode
prp291
•
0
@prp291-9622
Last seen 7.8 years ago
Hi I am using the latest version of CRISPRseek (CRISPRseek_1.15.1). I used following script to search the gRNA without any off-target analysis
results <- offTargetAnalysis(
inputFilePath,
findgRNAs=TRUE,
gRNA.name.prefix="gRNA",
PAM.size=3,
gRNA.size=20,
PAM="NGG",
gRNA.pattern = "^G", #gRNA start with G only
#gRNA.pattern= "^(?:(?!T{3,}).)+$", # three consecutive uracils in any position of a gRNA
#gRNA.pattern= "[ACG]{4,}.{3}$” , #avoid uracil in the last 4 positions of the guide sequence
min.score=0,
topN=1000,
findgRNAsWithREcutOnly = TRUE,
REpatternFile=system.file("extdata", "NEBenzymes.fa",package="CRISPRseek"),
minREpatternSize=6,
findPairedgRNAOnly = FALSE,
chromToSearch = "",
outputDir = outputDir,
overwrite = TRUE,
gRNAoutputName = "seq1gRNAs",
format="fasta",
exportAllgRNAs="fasta", #c("all", "fasta", "genbank", "no"),
fetchSequence=TRUE,
upstream=200,
downstream=200,
#upstream.search=0,
#downstream.search=0,
#baseBeforegRNA=4,
#baseAfterPAM=3,
useScore=TRUE,
overlap.gRNA.positions=c(17, 18),
useEfficacyFromInputSeq=TRUE,
outputUniqueREs=TRUE)
I used three different gRNA.pattern search but none of them produce the expected result. For example, gRNA.pattern = "^G" is also giving the gRNA starts with other than G. Thanks
Thanks. I am using this FASTA sequence to search the gRNA.
>test
ATGACGGAAAATTCAAAAGTATAACAATATAATTTTCAGGAAAATCATACTAATGATGTTCAAGGATTATTGAGTTTGTATGAAGCAGCACATATGAGAGTGCACGACGAGGAAATTCTAGAAGAAGCTCTTATCTTTACCACGACTCATCTCGCCATCGTAATTCGCGAATTTTGGAGTCCGTGATCCCGAATTTGAGCAACTCGCTTAAGGTACAAGTTACTGAAGCCTTAAGCCATCCTATTCGCAAAGCTATACCAAGGGTGGGAGCAAGGAAATACATACACATATATGAAAACATTGGAACACATAATGATTTACTTTTGAAATTTGCAAAGTTGGACTTCAACATGTTACAAAAGCTTCATCGAAAAGAGCTTAACGAGCTAACAAGGTACATCTACTATTCTTGTCATCTTCATAAGAGAGATGTGTACGCAGATCTTACCCTACCTTATAGAGGTAGAAATGTTGTTCCCGATTTATGGTACAATCAGACCTCTCTATAAAACACATCCTTTATAACAACAGTTCACTATAACGGTCAAGTTTTCTTTAAAATCAATCTTTTATGTTACCAAATATTTTTGAAAGAAACGTGACTATTATAGAGAGGTTTGACTGTAACTCGCGCTAATTAATAACACCTAAAGTTTAAGTATGTTAATGTTGTTATGATATCTATAGCTGGTGGAAAGATTTGGATCGTGCAAACAAATATGGATACACAAATCCCAAATCCAAAGTTAAAGACTTGATTGCTTCGTTGTTTGTCGAATCTGT
Thanks for help. I just modified my script like this to get the minimum free en-ergy of the secondary structure of gRNA with gRNA backbone but I didn't get anything. Actually Summary file is also not generated in the end. Can you check whether it is also a kind of bug.
results <- offTargetAnalysis( inputFilePath, findgRNAs=TRUE, gRNA.name.prefix="gRNA", PAM.size=3, gRNA.size=20, PAM="NGG", #gRNA.pattern = "^G", gRNA.pattern= "^G(?:(?!T{3,}).)+$", # Start with G and avoid three consecutive uracils in any position of a gRNA #gRNA.pattern= “[ACG]{4,}.{3}$”, #avoid uracil in the last 4 positions of the guide sequence min.score=0, topN=1000, findgRNAsWithREcutOnly = TRUE, REpatternFile=system.file("extdata", "NEBenzymes.fa",package="CRISPRseek"), minREpatternSize=6, findPairedgRNAOnly = FALSE, chromToSearch = "", outputDir = outputDir, overwrite = TRUE, gRNAoutputName = "seq1gRNAs", format="fasta", exportAllgRNAs="fasta", #c("all", "fasta", "genbank", "no"), fetchSequence=TRUE, upstream=200, downstream=200, #upstream.search=0, #downstream.search=0, #baseBeforegRNA=4, #baseAfterPAM=3, useScore=TRUE, overlap.gRNA.positions=c(17, 18), useEfficacyFromInputSeq=TRUE, outputUniqueREs=TRUE, foldgRNAs=TRUE, gRNA.backbone="GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUU", temperature=37, scoring.method="CFDscore", subPAM.activity = hash( AA = 0, AC = 0, AG = 0.259259259, AT = 0, CA = 0, CC = 0, CG = 0.107142857, CT = 0, GA = 0.069444444, GC = 0.022222222, GG = 1, GT = 0.016129032, TA = 0, TC = 0, TG = 0.038961039, TT = 0))Thanks for your help.